Microfluidic chip for analysis of cell motility and methods for using same

ABSTRACT

The present invention describes an integrated apparatus that enables identification of migratory cells directly from a specimen. The apparatus only requires a small number of cells to perform an assay and includes novel topographic features which can reliably differentiate between migratory and non-migratory cell populations in a sample. Both the spontaneous and chemotactic migration of cancer cells may be measured to distinguish between subpopulations within a tumor sample. The migratory cells identified using the apparatus and methods of the present invention may be separated and further analyzed to distinguish factors promoting metastasis within the population. Cells in the apparatus can be treated with chemotherapeutic or other agents to determine drug strategies to most strongly inhibit migration. The use of optically transparent materials in some embodiments allows a wide range of imaging techniques to be used for in situ imaging of migratory and non-migratory cells in the apparatus. The apparatus and methods of the present invention are useful for predicting the metastatic propensity of tumor cells and selecting optimal drugs for personalized therapies.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/906,055, filed Jan. 19, 2016, which is a 35 U.S.C. § 371U.S. national entry of International Application PCT/US2014/046639,having an international filing date of Jul. 15, 2014, which claims thebenefit of U.S. Provisional Application No. 61/847,187, filed on Jul.17, 2013, the content of each of the aforementioned applications isherein incorporated by reference in their entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant no.NCI-U54-CA143868 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Personalized medical plans aiming to limit metastasis are difficult todevelop. The current state of the art requires the expansion of humancancers in immunodeficient mice before the cancers can be subjected todrug screenings. It is known that metastatic subpopulations of cancercells have heighted motility which is linked to aggressiveness andinvasiveness of the cancer. The ability to identify such a subpopulationof cells in a tumor of a patient would be useful in classifying theaggressiveness or metastatic potential of the cancer in the subject, andwould also be useful in identifying optimal courses of treatment anddetermining whether the treatment was effective.

There currently exists no means for determining the motility of a cellor subpopulation of cells in a sample which is low cost, highthroughput, and easy to operate.

SUMMARY OF THE INVENTION

In accordance with an embodiment, the present invention provides anapparatus for analysis of cellular motility in a sample comprising: a) asubstrate in the form of a chip having at least a first and secondlayer, wherein the first layer is a fluid layer having at least a firstand second channel adjacent to each other on the fluid layer, the firstand second channel each having an inlet end and an outlet end, the firstchannel comprises one or more inlets, each inlet having a reservoirwhich communicates with the inlet end of the first channel, the firstchannel also comprises one or more outlets, each outlet having areservoir which communicates with the outlet end of the first channel,the second channel comprises an inlet having a reservoir whichcommunicates with the inlet end of the second channel and also comprisesan outlet having a reservoir which communicates with the outlet end ofthe second channel, b) the first and second channels are incommunication with each other through a plurality of migration channels,the migration channels comprise at least one inlet end and one or moreoutlet ends, each inlet end of the migration channels are incommunication with the second channel, and each of the one or moreoutlet ends of the migration channels are in communication with thefirst channel, and wherein the migration channels have a narrower widthand lesser height than either the first and second channels, and c) thesecond layer is a coverslip or polymer layer comprising a transparentsubstrate, which is bonded to the first layer to create a liquid seal.

In some embodiments, channels may then be functionalized with a proteinor cellular adhesion ligand to promote cell adhesion.

In accordance with another embodiment, the present invention provides amethod for analysis of the motility of a population of cells in a samplecomprising: a) adding to the inlet reservoir of the second channel ofthe apparatus described above, an aliquot of a suspension of apopulation of cells from the sample; b) incubating the cells for aperiod time to allow the cells to fill the second channel; c) removingany remaining cell suspension from the reservoir of the second channeland washing the inlet of the second channel; d) adding cell media to theone or more reservoirs of the one or more inlets of the first and secondchannels; e) imaging the cells in the apparatus for a period of time;and f) comparing the images of the cells in the apparatus over time andidentifying a cell or subpopulation of cells in the sample as migratorywhen the cell or subpopulation of cells migrates to the bifurcation ofany of the migratory channels of the apparatus.

In accordance with a further embodiment, the present invention providesa method for identifying the metastatic propensity of a cancer cell orpopulation of cells in a sample comprising: a) adding to the inletreservoir of the second channel of the apparatus described above, analiquot of a suspension of a population of cancer cells from the sample;b) incubating the cells for a period time to allow the cells to fill thesecond channel; c) removing any remaining cell suspension from thereservoir of the second channel and washing the inlet of the secondchannel; d) adding cell media to the one or more reservoirs of the oneor more inlets of the first and second channels; e) imaging the cells inthe apparatus for a period of time; and f) comparing the images of thecells in the apparatus over time and identifying a cell or subpopulationof cells in the sample as having a metastatic propensity when the cellor subpopulation of cells migrates to the bifurcation of any of themigratory channels of the apparatus.

In accordance with yet another embodiment, the present inventionprovides a use of the methods described above, to diagnose and/or treata disease or condition in a subject.

In accordance with an embodiment, the present invention provides amethod for selecting a molecule which modulates the motility of a cellor population of cells in a sample comprising: a) adding to the inletreservoir of the second channel of the apparatus described above, analiquot of a suspension of a population of cells from the sample; b)incubating the cells for a period time to allow the cells to fill thesecond channel; c) removing any remaining cell suspension from thereservoir of the second channel and washing the inlet of the secondchannel; d) adding cell media containing the a molecule of interest tothe one or more reservoirs of the one or more inlets of the first andsecond channels; e) imaging the cells in the apparatus for a period oftime; f) imaging the cells in the apparatus for a period of time; and g)comparing the images of the cells in the apparatus over time andcomparing the number and/or extent of migration of the cell orsubpopulation of cells to the number and/or extent of migration of thecell or subpopulation of cells migrating in the absence of the moleculeof interest; wherein if the number and/or extent of migration of thecell or subpopulation of cells off) is significantly greater or lesserthan that of the cell or subpopulation of cells migration in the absenceof the molecule of interest, determining that the molecule of interestmodulates the migration of the cell or subpopulation of cells.

In some embodiments, a plurality of molecules can be tested and resultscompared to select the most effective migration modulator.

In accordance with an embodiment, the present invention provides amethod for identifying a molecule which modulates the motility of a cellor population of cells in a sample comprising: a) adding to the inletreservoir of the second channel of the apparatus described above, analiquot of a suspension of a population of cells from the sample; b)incubating the cells for a period time to allow the cells to fill thesecond channel; c) removing any remaining cell suspension from thereservoir of the second channel and washing the inlet of the secondchannel; d) adding cell media to the one or more reservoirs of the oneor more inlets of the first and second channels; e) imaging the cells inthe apparatus for a period of time; f) comparing the images of the cellsin the apparatus over time and identifying a cell or subpopulation ofcells in the sample as migratory when the cell or subpopulation of cellsmigrates to the bifurcation of any of the migratory channels of theapparatus; g) isolating the migratory cells off) and optionally,expanding the population of migratory cells; h) repeating steps a)-c)with the isolated cells of g); i) adding cell media containing themolecule of interest to the one or more reservoirs of the one or moreinlets of the first and second channels; j) imaging the cells in theapparatus for a period of time; and k) comparing the images of the cellsin the apparatus over time and comparing the number and/or extent ofmigration of the cell or subpopulation of cells to the number and/orextent of migration of the cell or subpopulation of cells off) or thecell or subpopulation of cells migrating in the absence of the moleculeof interest; wherein if the number and/or extent of migration of thecell or subpopulation of cells of k) is significantly greater or lesserthan that of f), determining that the molecule of interest modulates themigration of the cell or subpopulation of cells.

In accordance with a further embodiment the present invention provides ause of the methods described above, to identify an optimal therapeuticagent for treatment of a subject. An optimal therapeutic agent woulddecrease migration of a cell or subpopulation of cells from the subject,as determined using the methods described above, and inhibit metastasisin the subject. Multiple devices can be operated in parallel to screen anumber of therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an embodiment of the apparatus of the presentinvention. (1A) A schematic of device, showing overall design of anembodiment used in the methods disclosed herein. Inset shows details ofY-shaped microchannels. (1B) Schematic of completed PDMS device bondedto glass coverslip. (1C) Schematic of cells seeded at channel bases.(1D) Phase contrast image of MDA-MB-231 cells migrating in 200 μm-longY-shaped microchannels.

FIG. 2 illustrates the migration of migratory and non-migratoryMDA-MB-231 cells within the device. (2A) Representative image ofmigratory cell in 3 μm-wide branch channel. (2B) Representative image ofnon-migratory cell within base channel of device. Migratory cells movedwithin the microchannels with significantly greater average speed (2C)and chemotactic index (2D). (2E) Ellipses fit to the cell outlines hadmajor axes highly aligned with the base channel in 89% of the migratorycells but only 52% of the non-migratory cells. (2F) Migratory cells weresignificantly more elongated than non-migratory cells, as measured bythe circularity of the cell outline. *, p<0.05.

FIG. 3 shows fluorescent on-chip imaging of F-actin and Rho GTPases.MDA-MB-231 ells within the microchannels were fixed and stained for (3A,D) F-actin, (3 B,E) Rac1, or (3 C,F) Cdc42. Panels 3A-C show cellsentering the 20 μm-wide branch channel. Panels 3D-F show cells enteringor migrating within the 3 μm-wide branch channel. Non-migratory cellsare shown at the 20 μm-wide channel bases in panels B and D (arrows).

FIG. 4 depicts how migratory MDA-MB-231 cells are contact guided at themicrochannel bifurcation. (4A) Representative cell tracks of migratorycells. Cells migrate predominantly up one channel wall and continue tofollow that wall at the bifurcation as they enter a branch channel. (4B)Percentage of migratory cells that were contact guided to the 3 μm-wideand 20 μm-wide branches.

FIG. 5 illustrates that PI3K inhibition promotes MDA-MB-231 cellmigration and contact guidance within the microchannels. Representativetracks of (5A) control and (5B) PI3K inhibited cells migrating within200 μm-long microchannels. PI3K inhibition with 10 μM LY294002 increasedthe percentage of cells that were migratory and that were contactguided. (5C) The average speed of control and LY294002-treated cells wasthe same. (5D) The chemotactic index of LY294002-treated cells wasgreater than that of control cells.

FIG. 6 depicts the extraction of migratory A375 cells from amicrochannel cell migration device of the present invention. (6A)Trypsinized cells flowed in the upper medium line without entering themicrochannels. Arrow indicates cell that has been completely detachedfrom the device. (6B) Detached cells flowed to the upper medium outletwell. Arrow indicates cell entering the well. (C) Extracted A375 cellswere expanded using standard cell culture techniques for 20 days andassayed for surface protein expression levels of the cancer stem cellmarker CD271 using flow cytometry. A375 cells that had migrated throughthe device and been expanded (blue) exhibited higher surface proteinexpression levels of CD271 than the bulk A375 cell population (green).

FIG. 7 depicts the isolation of migration MDA-MB-231 cells from amigration device of the present invention. (7A) Migratory cells thatexited the microchannels are shown prior to (left panel) and following(right panel) extraction from the device. Note that the position of thenonmigratory cells and cells seeded at the entrances to the channels isthe same before and after extraction of migratory cells. (7B) Orthotopicinjection of migratory cells but not control cells to the mammary fatpad of immunodeficient mice resulted in the formation of metastases(arrows).

FIG. 8 enumerates the percentage of migratory cells as determined usingthe present invention for a panel of breast epithelial or cancer celllines. Nonmetastatic cell lines were not migratory in the device (lessthan or equal to 1% of cells entering the migration channels reached thebranch channels following the bifurcation). In contrast, metastatic celllines contained a migratory subpopulation (more than 10% of the cellsfrom each cell line scored as migratory in the present invention).

FIG. 9 demonstrates the differential response of triple-negative breastcancer (TNBC) cell lines to an example pharmacological agent.MDA-MB-436, MDA-MB-231, Bt549, and Hs578t cells were treated with 10 μMLY294002, an inhibitor of PI3K, or the appropriate control. Migration ofMDA-MB-231 cells in the invention increased, while migration of Bt549cells decreased. A similar percentage of MDA-MB-436 and Hs578t cellswere migratory in the presence or absence of the inhibitor.

FIG. 10 depicts a representative sample of imaging techniques that canbe used to assess migration in the present invention. (10A) Lens-freeholography enables a wide field of view to be captured in a singleimage. Inset shows digitally zoomed image, with cells clearly visible inmigration channels. (10B) Phase contrast microscopy (10 x objective)image of migration channels with cells seeded at channel entrances.(10C) Combined phase contrast and fluorescence microscopy of cellswithin migration channels. A subset of cells was tagged with afluorescent marker and appears green in the image.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes an integrated apparatus that enablesidentification of migratory cells directly from a specimen. Theapparatus only requires a small number of cells to perform an assay. Theapparatus of the present invention includes novel topographic featureswhich can reliably differentiate between migratory and non-migratorycell populations in a sample. Furthermore, in some embodiments, both thespontaneous and chemotactic migration of cancer cells may be measured todistinguish between subpopulations within a tumor sample. The migratorycells identified using the apparatus and methods of the presentinvention may be separated and further analyzed to distinguish factorspromoting metastasis within the population. Cells in the apparatus canbe treated with chemotherapeutic or other agents to determine drugstrategies to most strongly inhibit migration. The use of opticallytransparent materials in some embodiments allows a wide range of imagingtechniques to be used for in situ imaging of migratory and non-migratorycells in the apparatus. The apparatus and methods of the presentinvention are useful for predicting the metastatic propensity of tumorcells and selecting optimal drugs for personalized therapies.

In an embodiment, the apparatus comprises a substrate in the form of achip having a plurality of layers. In one embodiment, the chip comprisesa fluid layer and a coverslip layer, which are fused together at finalassembly of the apparatus. The fluid layer is composed of a plurality ofchannels having at least one or more inlets and outlets.

In an embodiment, the fluid layer of the apparatus of the presentinvention comprises at least two channels, each having at least one ormore inlets which communicate with a reservoir, and each channel alsohaving at least one or more outlets which communicate with a reservoir.The channels can have any dimension within the limits of the depth ofthe substrate. In some embodiments, the channels can have dimensions ofabout 30 μm to about 100 μm in height and about 100 μm to about 400 μmwide.

Within the fluid layer of the substrate, in some embodiments, there areat least two channels, a first channel, also termed “a medium channel”which can be filled with any type of biological media or solvent. Thereis also at least a second channel, also termed “a cell channel” whichcan be filled with any type of biological media or solvent that containsa sample of cells to be assayed. In an embodiment, the first and secondchannels are disposed in proximity to each other and are parallel intoat least a portion of the two channels in the fluid layer of thesubstrate. The first and second channels have at least one inlet portionwhich can be the same or have a smaller dimension than the main portionof the first and second channel. Each inlet portion is connected to theinlet end of the first and second channel and communicates with thechannels. Each inlet portion is also in communication with a reservoirwherein media or fluid can be introduced into the inlet of the channel.

The first and second channels have at least one outlet portion which isthe same dimension as the main portion of the first and second channel.Each outlet portion is connected to the outlet end of the first andsecond channel and communicates with the channels. Each outlet portionis also in communication with a reservoir wherein media or fluid can bedirected to or removed from the channel.

In one or more embodiments, a novel aspect of the apparatus of thepresent invention is located in the migratory channel portion of theapparatus. In an embodiment, the migratory channel portion is an areawhere the first channel and second channel are in proximity to eachother and are connected by a plurality of migratory channels having atleast one inlet and at least one or more outlets. The migratorychannels, in some embodiments, are bifurcated at a point distal from theinlet portion of the migratory channel. In some embodiments, thebifurcation results in two outlet portions of the migratory channelwhich communicate with the media channel. These channels aresignificantly reduced in size, for example, by approximately by a factorof 10, so as to allow one cell body at a time to enter the migratorychannel from the cell channel. For example, in an embodiment, the mainportion of the first and second channels has a width of about 400 μm anda height of about 50 μm, whereas the migratory channels have an inletportion which communicates with the second channel and has a width ofabout 20 μm and a height of about 10 μm. In some embodiments, themigratory channels can have dimensions of width of about 3 μm to about50 μm, and a height of about 4 μm to about 15 μm. The one or more outletportions of the migratory channels can have the same or different widthsthan the inlet portion of the migratory channel. The bifurcation angleof the migratory channels is about 30° to 70° from the horizontal, whichis defined as the long axis of the first and second channels. It is inthese migratory channels that the cells in the sample are assayed fortheir ability to transverse the migratory channels and their speed,physical and biochemical characteristics can be measured.

Referring now to FIG. 1A which depicts an embodiment of the apparatus ofthe present invention, the fluid layer of the substrate is showngenerally as (1) and is composed of a polydimethylsiloxane (PDMS) chipmolded from a negative replica on a silicon wafer on whichphotolithography has been used to create a plurality of channels. Afirst channel (2), which has an inlet portion (3) and an outlet portion(4). The inlet portion is in communication with three inlets (5), termed“medium inlets” which are channels in the substrate that communicatebetween the inlet portion (3) and an inlet reservoir (6). The outletportion (4) is in communication with an outlet reservoir (7), termed“upper outlet.” On the fluid layer is also disposed a second channel(8), termed “cell channel” which has an inlet portion (9) and an outletportion (10). The inlet portion (9) of the second channel is incommunication with an inlet reservoir (11), termed “cell inlet.” Theoutlet portion (10) of the second channel is in communication with anoutlet reservoir (12), termed “lower outlet.” In some embodiments, theinlet and outlet reservoirs are punched into the substrate of thecoverslip layer having a circular shape and a diameter of about 6 mm,although that is only limited by the size of the volume required and thespace available on the substrate.

On the fluid layer there is a migratory channel portion (13) which is aregion between the first channel (2) and second channel (3) that has aplurality of migratory channels (14) which communicate with the firstand second channels. As seen on the exploded inset in FIG. 1A, themigratory channels (14) have an inlet end (15) which communicates withthe second channel (3), and two or more outlet ends (16), which are incommunication with the first channel (2). The two or more outlet ends(16) are the result of a bifurcation (17) of the migratory channel and apoint distal from the inlet end (15) of the migratory channel. In anembodiment, there are about 16 migratory channels (14) which connect thefirst (2) and second (3) channels in the migratory channel portion (13)of the fluid layer.

In an alternative embodiment, there are about 240 migratory channels(14) which connect the first (2) and second (3) channels in themigratory channel portion (13) of the fluid layer.

In some embodiments, the first and second channels are between about 10to about 50 mm in length, and have a height/depth of between about 30 toabout 100 μm, and a width of about 100 to about 400 μm. In someembodiments, the inlets for the first and second channels have a lengthof between 2 to about 10 mm, a height/depth of between about 30 to about100 μm, and a width of about 50 to about 400 μm. In some embodiments,the migratory channels have a length of between about 200 to 400 μm, aheight/depth of between about 4 to about 15 μm, and a width betweenabout 3 to about 50 μm.

As seen in FIG. 1B, the apparatus has a coverslip layer (18) which inwhole, or in part, is made of an optically transparent substrate. Anyoptically substrate which is compatible with the fluid layer substratecan be used. In an embodiment, the coverslip layer is formed of glass.It will be understood by those of ordinary skill that the portion of thecoverslip layer that is optically transparent will allow visualizationand imaging of the cells in the apparatus in real time. The coverslip isbonded using a variety of known means. In an embodiment, the coverslipis bonded to the fluid layer via plasma treatment of about 18 W for asufficient time, for example, about 2 minutes.

Before use, the fluid layer channels are all treated with 20 μg/mlcollagen, such as rat tail collagen type I, for about an hour at 37° C.,and then the channels are washed with PBS or similar buffer. In someembodiments, other extracellular proteins, such as fibronectin, VCAM-1,hyaluronic acid, or gelatin, are used.

The apparatus can be used for a variety of assays to detect and quantifythe micromechanical, morphological, and molecular signatures ofmigratory and non-migratory cells in the device.

Generally, operation of the apparatus comprises a first wash of thefirst channel and media inlet reservoirs (6) with a medium free buffersuch as DPBS. This is followed by seeding of cells of interest from asample. Cells of interest are seeded or introduced into the cell inletreservoir (11) of the second channel. In some embodiments, the cells aretrypsinized and suspended in serum free medium at a concentration ofabout 1×10⁵ to about 5×10⁶ cells/ml. In an embodiment, the cells aresuspended at a concentration of about 2×10⁶ cells/ml. About a 50 μlaliquot of the cell suspension is introduced into the cell inletreservoir (11), and the cells are incubated a 37° C. for a timesufficient to allow the cells to seed at the base of the migratorychannels (14), for example, about 2 to about 30 minutes, preferablybetween about 5 to about 10 minutes. In an embodiment, about 10 to 50 μlof a suitable biological medium or buffer are introduced to thelowermost medium inlet reservoir (6) to prevent convective flow of cellsthrough the migratory channels (14). Any remaining cells in the cellinlet reservoir (11) are then removed. Cell seeding is followed byintroduction of a suitable biological medium or buffer into the mediachannel of the apparatus via the medium inlet reservoirs (6) and theflow is in the direction of the upper outlet (FIG. 1A). A suitablebiological medium or buffer is also introduced into the cell inletreservoir (11) of the second channel. The apparatus can be manipulatedto either induce a chemoattractant gradient across the migratorychannels, or not to have any chemoattractant gradient. When inducing agradient, biological medium or buffer containing the chemoattractant isintroduced into the uppermost medium inlet reservoir (6), withbiological medium or buffer without the chemoattractant introduced intothe remaining medium inlet reservoirs (6), and into the cell inletreservoir (11) of the second channel. This creates a chemoattractantgradient across the migratory channels (14). If no gradient is desired,the medium inlet reservoirs (6) and cell inlet reservoir (11) are filledwith the same biological medium or buffer. In some embodiments, otherbiologically active compounds or molecules can be added to the cellsuspension when the cells are introduced into the apparatus, or afterthe cells have been seeded, to perform a variety of experiments.

In operation, the apparatus is placed in a temperature and CO₂controlled incubator, imaging chamber or stage type device, to which ismounted an imaging system. In some embodiments, the migrating cells areimaged at 10× magnification using a phase contrast or other opticalarrangement and images are taken at periodic intervals and saved oncomputer or other electronic storage media for up to about 16 hours. Itwill be understood by those of ordinary skill that the type ofmicroscopic imaging equipment can vary and can include any known systemsor apparatus which can image cells using any type of electromagneticradiation. Imaging systems include, but are not limited to, phasecontrast, brightfield, differential interference contrast, fluorescence,and confocal microscopy and in-line holography.

In accordance with one or more embodiments, the operation of theapparatus and methods of the present invention can be performed withoutthe need of external pumps or valves, and the function is driven bygravity and the topography of the channels in the apparatus. However, itis contemplated that alternative embodiments of the invention couldencompass external pumps or valves depending on the function desired,and is well within the ability of the skilled artisan.

In a preferred embodiment, the apparatus and methods of the presentinvention include methods for prognostic purposes in which migration ofcancer cells to one of the bifurcation branches of the migrationchannels is associated with a diseased state. In this embodiment,migration in the device serves as a companion diagnostic with othermethods of cancer diagnosis. A high migration score indicates that thespecific cancer tested has a high propensity to metastasize andindicates that aggressive treatment should be undertaken.

It is also contemplated that the apparatus of the present invention canbe used to isolate or separate a cell or subpopulation of cells from acollection of cells in a sample. In accordance with an embodiment, cellscan be separated from the device by means of trypsinization orchelation, which allows the cells to detach from the channel walls. Forexample, trypsin or EDTA can be introduced into all of the inletreservoirs. The cells detach and the flow of the apparatus is such thatthe cells that have migrated through the migratory channels will flowthrough the first channel and move into the upper outlet reservoir (FIG.2A).

In some embodiments, the migratory cells may be isolated from theapparatus and subjected to genomic or proteomic analysis. Such analysisincludes, but is not limited to, analysis of gene expression levelsusing quantitative real-time polymerase chain reaction and of surfaceprotein expression levels using flow cytometry.

In accordance with an embodiment of the present invention, it will beunderstood that the term “biological sample” or “biological fluid”includes, but is not limited to, any quantity of cells from a living orformerly living subject. Such cells include, but are not limited to,blood, bone, bone marrow, T-cells, B-cells, fibroblasts, chondrocytes,synovial macrophages, endothelial cells, tumor associated cells, andskin cells.

As used herein, the term “subject” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Logomorpha, such as rabbits. It ispreferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

The solid substrate used to make the apparatus of the present inventionmay be any suitable material. Representative examples of substratesinclude glass and modified or functionalized glass, plastics (includingacrylics, polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon, etc.),polysaccharides, nylon or nitrocellulose, resins, silica or silica-basedmaterials including silicon and modified silicon including PDMS, carbon,metals, inorganic glasses and plastics. In a preferred embodiment, thematerial used in the substrate is modified silicon.

In some embodiments, the apparatus and methods used are methods ofdiagnosis and the migration of cells is associated with a diseasedstate. In one preferred embodiment, the migration of cells is associatedwith cancer, such as prostate cancer, melanoma, bladder cancer, breastcancer, lymphoma, ovarian cancer, lung cancer, colorectal cancer or headand neck cancer. In other preferred embodiments, migration of cells isassociated with an immunological disorder; inflammation; rheumatoidarthritis; cystic fibrosis; or an infection, for example, a viral orbacterial infection. In other embodiments, the apparatus and methodsused are methods of monitoring prognosis and the migration of cells isassociated with the prognosis of a disease.

In yet another embodiment, the apparatus and methods used are formonitoring drug treatment and the migration of cells is associated withthe drug treatment. In particular, the apparatus and methods used are(e.g., analysis of migration of cells) for the selection ofpopulation-oriented drug treatments and/or in prospective studies forselection of dosing, for activity monitoring and/or for determiningefficacy endpoints. In this embodiment, decreased migration uponapplication of a particular biologically active molecule indicates thatthat molecule effectively inhibits the movement of migratory cells.

The diagnosis can be carried out in a person with or thought to have adisease or condition. The diagnosis can also be carried out in a personthought to be at risk for a disease or condition. “A person at risk” isone that has either a genetic predisposition to have the disease orcondition or is one that has been exposed to a factor that couldincrease his/her risk of developing the disease or condition.

Detection of cancers at an early stage is crucial for its efficienttreatment. Despite advances in diagnostic technologies, many cases ofcancer are not diagnosed and treated until the malignant cells haveinvaded the surrounding tissue or metastasized throughout the body.Although current diagnostic approaches have significantly contributed tothe detection of cancer, they still present problems in sensitivity andspecificity.

In accordance with one or more embodiments of the present invention, itwill be understood that the types of cancer diagnosis which may be made,using the apparatus and methods provided herein, is not necessarilylimited. For purposes herein, the cancer can be any cancer. As usedherein, the term “cancer” is meant any malignant growth or tumor causedby abnormal and uncontrolled cell division that may spread to otherparts of the body through the lymphatic system or the blood stream.

The cancer can be a metastatic cancer or a non-metastatic (e.g.,localized) cancer. As used herein, the term “metastatic cancer” refersto a cancer in which cells of the cancer have metastasized, e.g., thecancer is characterized by metastasis of a cancer cells. The metastasiscan be regional metastasis or distant metastasis, as described herein.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventiveapparatus and methods can provide any amount of any level of diagnosis,staging, screening, or other patient management, including treatment orprevention of cancer in a mammal

In accordance with the inventive apparatus and methods, the terms“cancers” or “tumors” also include but are not limited to adrenal glandcancer, biliary tract cancer; bladder cancer, brain cancer; breastcancer; cervical cancer; choriocarcinoma; colon cancer; endometrialcancer; esophageal cancer; extrahepatic bile duct cancer; gastriccancer; head and neck cancer; intraepithelial neoplasms; kidney cancer;leukemia; lymphomas; liver cancer; lung cancer (e.g. small cell andnon-small cell); melanoma; multiple myeloma; neuroblastomas; oralcancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer;sarcomas; skin cancer; small intestine cancer; testicular cancer;thyroid cancer; uterine cancer; urethral cancer and renal cancer, aswell as other carcinomas and sarcomas.

An “active molecule” and a “biologically active molecule” are usedinterchangeably herein to refer to a chemical or biological compoundthat induces a desired pharmacological and/or physiological effect,wherein the effect may be prophylactic or therapeutic. The terms alsoencompass pharmaceutically acceptable, pharmacologically activederivatives of those active agents specifically mentioned herein,including, but not limited to, salts, esters, amides, prodrugs, activemetabolites, analogs and the like. When the terms “active agent,”“pharmacologically active agent” and “drug” are used, then, it is to beunderstood that the invention includes the active agent per se as wellas pharmaceutically acceptable, pharmacologically active salts, esters,amides, prodrugs, metabolites, analogs etc.

As used herein, biologically active molecules which can be introducedinto the apparatus and used in the methods disclosed herein include, butare not limited to, dyes, including fluorescent, and NIRF dyes, enzymes,and enzyme linked dyes and markers, receptor antagonists or agonists,hormones, growth factors, autogenous bone marrow, antibiotics,antimicrobial agents, and antibodies. Non-limiting examples ofbiologically active agents include following: adrenergic blockingagents, anabolic agents, androgenic steroids, antacids, anti-asthmaticagents, anti-allergenic materials, anti-cholesterolemic and anti-lipidagents, anti-cholinergics and sympathomimetics, anti-coagulants,anti-convulsants, anti-diarrheal, anti-emetics, anti-hypertensiveagents, anti-infective agents, anti-inflammatory agents such assteroids, non-steroidal anti-inflammatory agents, anti-malarials,anti-manic agents, anti-nauseants, anti-neoplastic agents, anti-obesityagents, anti-parkinsonian agents, anti-pyretic and analgesic agents,anti-spasmodic agents, anti-thrombotic agents, anti-uricemic agents,anti-anginal agents, antihistamines, anti-tussives, appetitesuppressants, benzophenanthridine alkaloids, biologicals, cardioactiveagents, cerebral dilators, coronary dilators, decongestants, diuretics,diagnostic agents, erythropoietic agents, estrogens, expectorants,gastrointestinal sedatives, agents, hyperglycemic agents, hypnotics,hypoglycemic agents, ion exchange resins, laxatives, mineralsupplements, mitotics, mucolytic agents, growth factors, neuromusculardrugs, nutritional substances, peripheral vasodilators, progestationalagents, prostaglandins, psychic energizers, psychotropics, sedatives,stimulants, thyroid and anti-thyroid agents, tranquilizers, uterinerelaxants, vitamins, antigenic materials, and prodrugs.

When the biologically active molecule is a dye, the molecule is detectedby fluorescence imaging. The dyes may be emitters in the visible ornear-infrared (NIR) spectrum. Known dyes useful in the present inventioninclude carbocyanine, indocarbocyanine, oxacarbocyanine,thüicarbocyanine and merocyanine, polymethine, coumarine, rhodamine,xanthene, fluorescein, boron˜dipyrromethane (BODIPY), Cy5, Cy5.5, Cy7,VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680,AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752,Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680,HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS,ADS830WS, and ADS832WS.

The term “modulate,” as used herein means that in the presence of thebiologically active agent or molecule, the migratory ability of the cellor subpopulation of cells is up regulated or down regulated, such thatmigration level, or activity is greater than or less than that observedwhen compared to controls. For example, the term “modulate” can mean“inhibit,” but the use of the word “modulate” is not limited to thisdefinition.

The term “inhibit” as used herein, means that that in the presence ofthe biologically active agent or molecule, the migratory ability of thecell or subpopulation of cells is lowered or down regulated whencompared to controls.

EXAMPLES

Fabrication of an embodiment of the apparatus of the present invention:The microfluidic device consisted of “Y”-shaped microchannels, with a 20μm-wide feeder channel bifurcating to 20 μm-wide or 3 μm-wide branches,arrayed between mutually perpendicular cell seeding and cell outletchannels. Microchannels were of height H_(C)=10 μm and lengthL_(C)=200-400 μm and were spaced 50 μm apart.

The apparatus was fabricated using multilayer photolithography andreplica molding. Photolithography masks were designed using AutoCAD(Autodesk, McLean, Va.) and produced by the Photoplot Store (ColoradoSprings, Colo.). The master for the device contained a negative mold ofthe final device and was fabricated using SU-8 3010 positive photoresist(Microchem, Newton, Mass.). SU-8 3010 was spin coated (Single Wafer SpinProcessor, Model WS-400A-6NPP-LITE, Laurell Technologies, North Wales,Pa.) on a cleaned silicon wafer (University Wafer, South Boston, Mass.)to create a 10 μm-thick film. The film was soft baked on a hot plate andexposed to 170 mJ/cm2 of UV light energy through the chrome-on-glasslight field mask using an EVG620 mask aligner (EVG, Austria) to definethe microchannels. The wafer was baked, post-exposure, to cross link thepattern before development with SU-8 developer. Following development, a50 μm-thick SU-8 3025 film was spun onto the wafer and soft baked. Amask defining the medium feed lines was aligned with the channels, andthe photoresist was exposed to 250 mJ/cm2 of energy. The final masterwas developed, hard baked, and passivated with a fluorinated silane[(tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane] (Pfaltz &Bauer, Waterbury, Conn.) overnight in a vacuum desiccator.

Completed devices were molded from the SU-8 masters by pouringpolydimethylsiloxane (PDMS) (Sylgard® 184 Silicone Elastomer Kit, DowCorning, Midland, Mich.) at a 10:1 ratio of prepolymer:crosslinker overthe master, degassing, and curing at 85° C. for 2 hours. Devices werediced, and 6-mm inlet and outlet ports were punched in the PDMS fluidlayer. The devices and glass coverslips were cleaned with ethanol and DIwater and plasma treated for 2 minutes at 18 W (Harrick PDC-32G, HarrickPlasma, Ithaca, N.Y.). The device was bonded to the glass slide andcoated with 20 μg/ml rat tail collagen type I (BD, Franklin Lakes, N.J.,USA) for 1 hour at 37° C. Following coating, the channels were washedwith DPBS to prepare for cell seeding.

Description of an embodiment of the apparatus used in the examples ofthe present invention. The apparatus, termed a “Microchannel MigrationDevice,” comprises a plurality of Y-shaped microchannels arrayed betweencell seeding (second channel) and medium (first channel) lines (FIG.1A). The microchannels were designed such that 20 μm base channelsbifurcated to 20 μm and 3 μm branch channels at a 45° or 65° (from thehorizontal) angle (inset, FIG. 1A). With this device design, experimentscan be carried out with or without a chemoattractant gradient. If nogradient is desired, growth medium is placed in all four inlet wells,and the topography of the channels is the only driver of migration. Whenmedium containing a chemoattractant is placed in the uppermost mediuminlet well and medium without chemoattractant is placed in the cellinlet and bottom two medium inlet wells, a gradient is formed within themicrochannels to induce migration.

The particular embodiment of the device was formed by bonding a PDMSmold containing the microchannels and medium channels to a glasscoverslip (FIG. 1B). Cells were seeded at the bases of the microchannelsfollowing gravity-driven flow of suspended cells from the cell inletwell (shown schematically in FIG. 1C). Medium was placed in all inletwells following cell seeding. Importantly, cell seeding and migrationwere carried out without the need for external pumps or valves, and allflow was driven by gravity. Seeded cells migrated through the channelsover the course of the experiment (FIG. 1D).

Cell seeding and live cell migration experiments: Cells were grown toconfluency, trypsinized, and resuspended in serum-free medium at 2×10⁶cells/ml. 50 μl of cell suspension was added to the cell inlet well.Cells were incubated in the device for 5-10 minutes at 37° C. to allowinitial cell seeding at the base of “Y” channels. The cell suspensionwas then removed from the cell inlet port. The device was washed withDPBS before the addition of medium to the inlet ports of the device. Inselect experiments, PI3K activity was inhibited by the addition of 10 μMLY294002 in the medium through the entire course of migration. Themigration chamber was moved to a temperature- and CO₂-controlledstage-top live cell incubator (Okolab, Italy) mounted on the motorizedstage of an inverted Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan)with automated controls (NIS-Elements, Nikon). Migrating cells wereimaged with a 10×-magnification phase contrast objective every 10minutes for up to 16 hours.

Analysis of Cell Migration: Video files were exported to ImageJ foranalysis. All cells that entered the channel were tracked while fullyinside the channel and before reaching either end of the channel usingthe ImageJ MTrackJ plugin at 10 minute intervals. Cells were alsodynamically traced with the ImageJ polygon ROI capability at 30 minuteintervals. Dividing cells were not tracked.

Cell position data were used to calculate cell speed over each 10 minuteinterval, and these speeds were averaged to get an overall average speedfor each cell. Additionally, the chemotactic index, defined as the celldisplacement divided by the total distance travelled by the cell, wascalculated. Cell shape data were used to calculate cell circularity andfit elliptical angle using the Measure function in ImageJ. Statisticalsignificance was assessed with non-paired Student's t-test.

Cells were further defined as migratory or non-migratory. Migratorycells were defined as those cells which reached the bifurcation in theY-shaped microchannel; all other cells were defined as non-migratory.Migratory cells were then classified as contact guided or not contactguided. Cells were defined as contact guided if they continued to thebranch channel on the side of the base channel on which they weremigrating when the bifurcation was reached. Cells that switched walls inthe bifurcation region were classified as not contact guided.

Isolation of Migratory Cells: Cells that had migrated through and exitedthe channels were washed with a chelator (versene) prior to the additionof 0.25% trypsin to all inlet wells of the device. Hydrodynamicresistance to flow in the narrow microchannels prevented the backflow ofcells that had migrated through the microchannels back into themicrochannels. Detached cells flowed to the upper outlet well, werecollected in culture medium, and were plated in 96-well plates forexpansion. Expanded cells were analyzed for the presence of tumor stemcell markers (for example, CD44 or CD271).

Alternatively, ˜300 migratory cells were collected, suspended in 75 μlof DPBS, mixed with 75 μl of Matrigel, and injected to the mammary fatpad of an immunodificient mouse. An equal number of control cells thathad not migrated through the microchannels were collected and injectedin an identical manner. Mice were sacrificed 8 weeks post-injection, andthe lungs were histologically analyzed to detect metastases.

Example 1

Bifurcating Channels Allow Identification of Migratory Cells: Celltracking of all MDA-MB-231 cells within the channels revealed twodistinct subpopulations: migratory and non-migratory cells (FIGS. 2A,B).22±3% of human metastatic MDA-MB-231 breast cancer cells were migratory.Interestingly, this subpopulation correlates with the % of MDA-MB-231(28%) bearing the CD44⁺/CD24⁻ molecular signature⁵ that is used todefine breast cancer stem cells. Migratory cells, defined as those cellsreaching the branch channels, migrated more than twice as fast asnon-migratory cells (FIG. 2C). Migratory cells were also significantlymore directional. The chemotactic index of migratory cells increased to0.91 in comparison to a chemotactic index of 0.37 for nonmigratorycells.

Analysis of cell shape indicated that migratory cells were aligned withand elongated along the channel wall. The fit elliptical angle of a cellperfectly aligned along the wall was 90°. Although both migratory andnon-migratory cells had an average fit elliptical angle of 90°, 89% ofmigratory cells had fit elliptical angles within 10° of 90°, while only52% of non-migratory cells showed this high degree of alignment (FIG.2E). This directed migration was confirmed by analysis of migration inthe base channel. In that region of the microchannel, migratory cellschanged direction an average of 0.6 times, while non-migratory cellsaveraged 5.6 direction changes. Circularity, a shape factor thatdecreases as shapes become less circular, was also significantlydifferent between migratory and non-migratory cells. Migratory cellswere significantly more elongated as they migrated, with a circularityof 0.37. Non-migratory cells had an average circularity of 0.58 (FIG.2F). Additionally, the apparatus of the present invention was used foranalysis of cytoskeletal components and intracellular signals viafluorescence microscopy (FIG. 3 ). This was possible because the devicewas constructed of transparent materials. Migratory cells showedincreased localization of F-actin to the cell leading edge (FIG. 3A,D).Actin localization was not seen in non-migratory cells (rounded cell atbase of channel, FIG. 3D). Similarly, the Rho GTPases Rac1 and Cdc42were polarized in migratory cells, particularly when these cells reachedthe 3 μm-wide branch channel (FIG. 3B,C,E,F). Non-migratory cells didnot exhibit this polarization (for example, cell at channel base in FIG.3B).

Example 2

Contact Guidance Overcomes Steric Hindrance for Migratory Cells: Ofthose cells that were migratory, the vast majority moved preferentiallyalong one wall of the feeder channels and remaining polarized, with asignificantly lower number of changes in direction compared tonon-migratory cells. Representative cell tracks illustrating this trendare shown in FIG. 4A. Interestingly, migration direction at thebifurcation was not dependent on the width of the resultant branch, eventhough entering the 3 μm-wide branch required significant deformation ofthe cell body. Instead, cells continued to be polarized and movedreadily into the “branch” channel, regardless of the branch channelwidth (FIG. 4B). Thus, contact guidance dominated steric hindrance atthese channel widths for migratory cells and was likely the driver ofdirected migration for this subpopulation.

Example 3

PI3K Inhibition Promotes Spontaneous Migration of MDA-MB-231 Cells:There is evidence that PI3K signaling is required to stabilize nascentprotrusions. New protrusions away from the wall along which a cell ismigrating would discourage contact guidance. Therefore, it wasinvestigated whether inhibiting PI3K could promote contact guidance in200 μm-long microchannels.

Inhibition of PI3K signaling using the PI3K inhibitor LY294002 increasedthe migratory cell population in 200 μm-long microchannels from 25% to65% and the ratio of contact guided cells from 66% to 93% (FIG. 5A,B).PI3K inhibition did not impact overall cell speed, as control andLY294002-treated cells moved at the same average speed (FIG. 5C).However, cells in which PI3K signaling was inhibited moved with greaterdirectionality, as indicated by the higher chemotactic index for thesecells vs. control cells (FIG. 5D). This result is consistent with theexpected inhibition of nascent protrusions upon LY294002 treatment, asnew protrusions would be required for the cell to change direction.

Example 4

Device Design Allows Isolation of Migratory Cells: Furthercharacterization of migratory and non-migratory cells will provideimportant information on the nature of these cell populations. Forexample, we wish to characterize whether migratory cells show stem-likecharacteristics, retain high migratory potential over severalgenerations, or display differential gene expression in comparison tonon-migratory cells. To answer these questions, it will be necessary toisolate migratory cells from the device.

Proof-of-concept experiments were performed to isolate migratory cells.A375 cells migrated through straight microchannels toward a chemotacticcue. Trypsin was added to all inlet wells of the device and caused themigratory cells to become detached and flow to the upper medium outletwell (FIG. 6A). Resistance to flow through the narrow microchannelsprevented detached cells from flowing back into the microchannels.Migratory cells were collected in the upper medium outlet well (FIG. 6B)and plated in 96-well plates for expansion. Expanded cells were analyzedfor expression of the cancer stem cell marker CD271 using flowcytometry. Migratory A375 cells displayed increased expression of thismarker compared to cell populations from which the migratorysubpopulation had not been extracted (FIG. 6C).

Example 5

Migratory cells are more likely to cause metastasis upon orthotopicinjection in immunodeficient mice: MDA-MB-231 cells migrated through thedevice in the absence of a chemotactic cue. Trypsin was added to the allmedium inlet wells of the device and caused the migratory cells tobecome detached and flow to the upper medium outlet well (FIG. 7A).Resistance to flow through the narrow microchannels prevented detachedcells from flowing back into the microchannels (FIG. 7A; compare cellpositions in microchannels before and after removal of migratory cells).Approximately 300 migratory cells were collected in the upper mediumoutlet well. Trypsin was then added to the cell inlet well. Cells thathad not entered the channels flowed to the lower outlet well and werecollected. Approximately 300 of these control cells were collected.Migratory or control cells were suspended in 75 μl DPBS, mixed with 75μl Matrigel, and injected to the mammary fat pad of immunodeficientmice. Mice were sacrificed at 8 weeks post-injection. Histologicalanalysis of the lungs of these mice revealed that migratory cells causedlung metastasis, whereas control cells did not (FIG. 7B; arrows indicatemetastases).

Example 6

Observations using metastatic MDA-MB-231 breast cancer cells weregeneralized. A panel of cell lines was assayed in devices containing 400μm-long Y-shaped migration channels (FIG. 8 ). None of thenon-tumorigenic MCF10A breast epithelial cells or non-metastatic MCF-7breast cancer cells assayed were found to be migratory. Similarly, only1±1% of non-metastatic MDA-MB-468 breast cancer cells were migratory.Conversely, subpopulations of motile cells were found in metastaticK-Ras-overexpressing/obscurin-knockdown MCF10A cells (20%), metastaticBt-549 breast cancer cells (32±8%), metastatic MDA-MB-436 breast cancercells (13±7%), metastastic MDA-MB-231 breast cancer cells (22±3%),metastatic Hs578t breast cancer cells (20±7%), and metastatic A375melanoma cells (38±7%). The 38±7% of migratory human A375 melanoma cellsclosely matches the % of A375 cells expressing the cancer stem cellmarker CD271.

Example 7

Triple negative breast cancer cells display divergent responses topharmaceutical agents: A panel of triple-negative breast cancer (TNBC)cell lines were assayed in devices containing 400 μm-long Y-shapedmigration channels in the presence or absence of the PI3K inhibitorLY294002 (10 μM) (FIG. 9 ). PI3K inhibition did not affect thepercentage of migratory cells measured for MDA-MB-436 (13±7% migratoryfor control vs. 18±7% for treated) cells or Hs578t (20±7% migratory forcontrol vs. 15±2% migratory for treated) cells. PI3K inhibitionincreased migration of MDA-MB-231 (22±3% for control vs. 34±8% fortreated) cells. In contrast, PI3K inhibition reduced the migration ofBt549 (32±8% for control vs. 19±1% for treated) cells.

Example 8

Metastatic propensity assay is amenable to a wide range of imagingtechniques: Numerous imaging techniques were used to image an embodimentof the invention in which a PDMS fluidic layer is bonded to a glasscoverslip layer. Cells were imaged using lens-free holography (FIG.10A), which has an approximately 5 mm×5 mm field of view. Cells wereclearly visible in the channels upon digitally zooming in (inset, FIG.10A). Additionally, cells in the device were imaged using phase contrastmicroscopy (FIG. 10B) and a combination of phase contrast andfluorescence microscopy (FIG. 10C; a subset of cells was tagged with afluorescent marker and appears green in the image).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method for analysis of motility of apopulation of cells in a sample, the method comprising: a) adding asuspension of a population of cells from the sample to a chip having afluid layer having first and second channels, each of the first andsecond channels having a respective inlet end and a respective outletend, wherein the first channel comprises: one or more inlets in fluidcommunication with the inlet end of the first channel; and one or moreoutlets in fluid communication with the outlet end of the first channel,wherein the second channel comprises: an inlet in fluid communicationwith the inlet end of the second channel; and an outlet in fluidcommunication with the outlet of the second channel, wherein the firstand second channels of the fluid layer are in communication with eachother through at least one migration channel formed within the fluidlayer of the chip, wherein a first migration channel of the at least onemigration channel comprises: a single inlet end in fluid communicationwith the second channel of the fluid layer; first and second outlet endsin fluid communication with the first channel of the fluid layer; afirst body portion extending from the single inlet end toward the firstand second outlet ends; and first and second branch portions extending,respectively, from the first body portion to the first and second outletends, wherein the single inlet end of the first migration channel isconnected to the second channel of the fluid layer, wherein the firstand second outlet ends of the first migration channel are connected tothe first channel of the fluid layer, wherein the first body portion ofthe first migration channel and the first and second branch portions ofthe first migration channel are connected to one another at abifurcation of the first migration channel, wherein the first and secondbranch portions of the first migration channel have respective widths,wherein the width of the first branch portion of the first migrationchannel is different than the width of the second branch portion of thefirst migration channel; and wherein the suspension of the population ofcells is added from the sample to the inlet of the second channel; b)incubating the cells for a period time to allow the cells to fill thesecond channel; c) washing the inlet of the second channel; d) addingcell media to the one or more inlets of the first channel; e) imagingthe cells in the chip for a period of time, thereby producing one ormore images of the cells; and f) comparing the images of the cells inthe chip over time and identifying in the sample a migratory cell whenthe migratory cell or a subpopulation of migratory cells enters thesingle inlet end of the first migration channel and: (i) migrates to andenters the first or second branch portions of the first migrationchannel; (ii) exits the first or second outlet ends of the firstmigration channel; or (iii) both (i) and (ii).
 2. The method of claim 1,further comprising: g) identifying the migratory cell or thesubpopulation of migratory cells as contact-guided when the migratorycell or the subpopulation of migratory cells identified in step f)continues to migrate to the first and second branch portions on a sameside of the base portion of the first and second branch portions of thefirst migratory channel.
 3. The method of claim 1, further comprising:h) isolating the migratory cell or the subpopulation of migratory cellsfrom the chip.
 4. The method of claim 1, wherein the sample is obtainedfrom a subject.
 5. The method of claim 3, wherein the migratory cell orthe subpopulation of migratory cells is a cancer cell.
 6. The method ofclaim 1, wherein the first and second channels of the chip havedimensions (h×w) of 50 μm×400 μm.
 7. The method of claim 1, wherein thefirst channel of the chip has three inlets and one outlet.
 8. The methodof claim 1, wherein the second channel of the chip has one inlet and oneoutlet.
 9. The method of claim 1, wherein the single inlet end of thefirst migration channel has a dimension of 10 μm×20 μm.
 10. The methodof claim 1, wherein the first and second branch portions of the firstmigration channel have differing cross-sectional areas.
 11. The methodof claim 10, wherein the first and second branch portions of the firstmigration channel have respective dimensions of 10 μm×3 μm and 10 μm×20μm.
 12. The method of claim 1, wherein each migration channel of the atleast one migration channel of the chip has a length of 200 μm-400 μm.13. The method of claim 1, wherein the at least one migration channel ofthe chip comprises a plurality of migration channels that are spacedapart.
 14. The method of claim 1, wherein the chip comprises from 1 to400 migration channels.
 15. The method of claim 1, wherein the inlet andoutlet of the first and second channels each further comprise areservoir.
 16. The method of claim 1, wherein the imaging of cells isperformed using a method selected from the group consisting of phasecontrast; brightfield; differential interference contrast; fluorescence;and confocal microscopy and in-line holography.
 17. The method of claim1, wherein the period of time for imaging the cells is from 10 minutesto 16 hours.
 18. The method of claim 3, wherein the step of isolatingthe migratory cell or the subpopulation of migratory cells from the chipcomprises applying trypsin or a chelating agent to the chip.
 19. Amethod for analysis of motility of a population of cells in a sample,the method comprising: a) adding a suspension of a population of cellsfrom the sample to a chip having a fluid layer having first and secondchannels, each of the first and second channels having a respectiveinlet end and a respective outlet end, wherein the first channelcomprises: one or more inlets in fluid communication with the inlet endof the first channel; and one or more outlets in fluid communicationwith the outlet end of the first channel, wherein the second channelcomprises: an inlet in fluid communication with the inlet end of thesecond channel; and an outlet in fluid communication with the outlet ofthe second channel, wherein the first and second channels of the fluidlayer are in communication with each other through a plurality ofmigration channels formed within the fluid layer of the chip, whereinthe plurality of migration channels are spaced apart, and wherein eachmigration channel of the plurality of migration channels comprises: asingle inlet end in fluid communication with the second channel of thefluid layer; first and second outlet ends in fluid communication withthe first channel of the fluid layer; a first body portion extendingfrom the single inlet end toward the first and second outlet ends; andfirst and second branch portions extending, respectively, from the firstbody portion to the first and second outlet ends, wherein the first andsecond branch portions of a first migration channel of the plurality ofmigration channels have respective widths, wherein the width of thefirst branch portion of the first migration channel is different thanthe width of the second branch portion of the first migration channel;and wherein the suspension of the population of cells is added from thesample to the inlet of the second channel; b) incubating the cells for aperiod time to allow the cells to fill the second channel; c) washingthe inlet of the second channel; d) adding cell media to the one or moreinlets of the first channel; e) imaging the cells in the chip for aperiod of time, thereby producing one or more images of the cells; andf) comparing the images of the cells in the chip over time andidentifying in the sample a migratory cell when the migratory cell or asubpopulation of migratory cells enters the single inlet end of thefirst migration channel and: (i) migrates to and enters the first orsecond branch portions of the first migration channel; (ii) exits thefirst or second outlet ends of the first migration channel; or (iii)both (i) and (ii).
 20. The method of claim 19, wherein the population ofcells in the sample are cancer cells.